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The effect of experimental diabetes on drug induced responses in cardiac tissues of the rat McCullough, Ann Louise 1982

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THE EFFECT OF EXPERIMENTAL DIABETES ON DRUG INDUCED RESPONSES IN CARDIAC TISSUES OF THE RAT  by ANN LOUISE McCDLLOUGH  B.Sc,  The University of New Brunswick, 1979  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Division of Pharmacology and  Toxicology  o  of the Faculty of Pharmaceutical Sciences  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA August  c  1982  Ann Louise McCullough,  1982  In  presenting  requirements of  British  it  freely  agree for  this for  an  available  that  I  understood  that  financial  by  his  that  or  shall  be  her or  Date  vT-.  r  / —i  / —ir\ \  (Inn,  University  and  study.  I  copying  granted  by  publication be  allowed  Columbia  '  the  shall  of  /<?&-  the  the  of  of  this  It  this  without  make  further  head  representatives.  not  The U n i v e r s i t y of B r i t i s h 2075 W e s b r o o k P l a c e Vancouver, Canada V6T 1W5  at  of  Library  permission.  DeparJ-JfreTTt  fulfilment  the  extensive  may  copying  gain  degree  reference  for  purposes  or  partial  agree  for  permission  scholarly  in  advanced  Columbia,  department  for  thesis  thesis  of  my  is  thesis my  written  ABSTRACT The effect of experimental diabetes mellitus on the response of isolated cardiac tissues to the 6 adrenergic agonist d , l . isoproterenol and the cardiac glycoside ouabain was examined.  The relationship  between the duration of chemically induced diabetes and the to these drugs was also  response  investigated.  Basal developed tension was not different in control vs.diabetic p a p i l l a r y muscles 7 days or 70 days after the induction of  diabetes.  Tissues from 7 day diabetic animals responded to d , l isoproterenol in a similar manner to tissues from control an.imals at each drug dose. There was a non-significant depression in the response of both p a p i l l a r y muscles and l e f t a t r i a from 70 day diabetic r a t s . the dose-response  This trend was evident throughout  curves.  The basal rate of spontaneously beating isolated right a t r i a from 7 day STZ diabetic rats was s i g n i f i c a n t l y depressed compared to c o n t r o l , while that of alloxan diabetic animals was depressed to a smaller degree.  There was no difference in the maximum response of  these-tissues to d ,1 isoproterenol.  The basal rate was not different  in a t r i a from 70 day diabetic animals as compared to controls.  Tissues  from 70 day diabetic rats demonstrated a diminished response to d , l isoproterenol throughout the dose response curve however this depression was not s t a t i s t i c a l l y  significant.  There was no difference in tension development in l e f t a t r i a or p a p i l l a r y muscles at any time point. of diabetes  Seven days after the induction  both a t r i a and p a p i l l a r y muscles demonstrated a non-  s i g n i f i c a n t depression of the ouabain dose response curve.  ii  Papillary  muscles from 70 day diabetic animals displayed a s i g n i f i c a n t depression in these dose response curves at ouabain concentrations greater than 10  M.  Atria from six month diabetic rats demonstrated  s i g n i f i c a n t l y depressed curves 3 x 10"  5  at  concentrations greater than  M.  Ouabain produces a monophasic dose response curve in l e f t and a biphasic dose response curve in papillary muscles.  atria  Catecholamine  release does not appear to be involved in these responses. Chronic alloxan and streptozotocin diabetes produces changes in the myocardium of rats characterized by a diminished inotropic response to the cardiac glycoside ouabain.  This depression is not  accompanied by a s t a t i s t i c a l l y s i g n i f i c a n t decline in the maximum inotropic or chronotropic response to isoproterenol.  ii i  TABLE OF CONTENTS  PAGE  ABSTRACT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS  ii iv vi vii viii  INTRODUCTION  1  I  Diabetes Mellitus and Diabetic Cardiomyopathy 1.  Introduction  1  2.  Functional and Corresponding Morphological Changes in the Diabetic Myocardium  4  3.  Autonomic Neuropathy in Diabetes Mellitus  8  4.  Diabetes and Myocardial Metabolism  11  5.  Function of the Sarcoplasmic Reticulum in Diabetes  16  6.  The Effect of Diabetes Mellitus on the Contractile Units of the Myocardium  17  Summary  18  7.  II Cardiac Glycosides  and the Heart  19  1.  Introduction  2.  Role of (Na + K )-ATPase in the Positive Inotropic Action of Cardiac Glycosides  20  Low Dose Effects of Ouabain — Possible Biphasic Inotropic Response in Cardiac Tissues  26  Low S e n s i t i v i t y of Rat Hearts to Cardiac Glycosides.  30  3.  4.  5.  +  19 +  Insulin and Diabetes and the (Na ATPase Mediated Ouabain Response  III  1  Summary of Experimental Aims  MATERIALS AND METHODS  +  +  + K )32 34 35  1.  Induction of Diabetes  35  2. 3.  Maintenance of Animals Preparation of Isolated Tissues  35 36  iv  PAGE 4.  Preparation of Drugs  37  5.  Analysis of Serum  37  6.  Statistical  37  7.  Materials  Analysis  38  RESULTS  39  I  Detection o f Diabetes  39  II  Response to d , l Isoproterenol in Cardiac Tissues taken from Rats 7 or 70 Days after the Induction of Diabetes.  39  A.  Inotropic Responses  39  R.  Chronotropic Responses  45  III  Response to Ouabain in Cardiac Tissues 7 Days, 70 Days or 6 Months After the Induction of Diabetes., A. B.  IV  56  Effect of Timolol on Ouabain Dose Response Curves  56  Inotropic Responses to Ouabain in Left A t r i a and Papillary Muscles  56  Effect of Time and B Blockade on the Positive Inotropic Effect of Ouabain on Cardiac Tissues  60 74  DISCUSSION I  Inotropic Response to d , l Isoproterenol  74  II  Chronotropic Response to d , l Isoproterenol  75  III Response to Ouabain IV  V  Effect of Time and  768 Blockade on the Ouabain  Dose Response Curve  78  Conclusions  81  BIBLIOGRAPHY  82  v  LIST OF TABLES TABLE I  II  III  PAGE Inotropic Responses of Cardiac Tissues to d , l Isoproterenol 7 or 70 Days after the Induction of Diabetes  40  Chronotropic Responses of Right A t r i a to d , l Isoproterenol 7 or 70 Days after the Induction of Diabetes  50  Inotropic Responses of Cardiac Tissues to Ouabain at Various Times after the Induction of Diabetes  59  vi  LIST OF FIGURES  FIGURE  PAGE  1.  The S u b s t r a t e Supply o f the Normal Heart  13  2.  I n o t r o p i c Response o f P a p i l l a r y Muscles from C o n t r o l and 7 Day A l l o x a n D i a b e t i c Rats to d , l I s o p r o t e r e n o l  42  I n o t r o p i c Response o f L e f t P a p i l l a r y Muscles from C o n t r o l and 70 Day D i a b e t i c Rats t o d , l I s o p r o t e r e n o l  44  I n o t r o p i c Response o f L e f t A t r i a l T i s s u e s from C o n t r o l and 7 Day D i a b e t i c Rats to d-,1 I s o p r o t e r e n o l  47  I n o t r o p i c Response o f L e f t A t r i a from C o n t r o l Day D i a b e t i c Rats t o d , l I s o p r o t e r e n o l  49  3.  4.  5. 6. 7. 8. 9. 10. 11. 12.  and 70  C h r o n o t r o p i c Response o f R i g h t A t r i a from C o n t r o l 7 Day D i a b e t i c Rats to d , l I s o p r o t e r e n o l .  and  C h r o n o t r o p i c Response o f R i g h t A t r i a from C o n t r o l 70 Day D i a b e t i c Rats to d , l I s o p r o t e r e n o l  and  E f f e c t o f 3 B l o c k a d e i n the P o s i t i v e I n o t r o p i c to Ouabain i n L e f t A t r i a from Rats  52 55  Response 58  I n o t r o p i c Response o f L e f t A t r i a from C o n t r o l D i a b e t i c Rats to Ouabain  and 7 Day  I n o t r o p i c Response o f L e f t A t r i a from C o n t r o l D i a b e t i c Rats to Ouabain  and 6 Month  62 64  I n o t r o p i c Response o f P a p i l l a r y Muscles from C o n t r o l 7 Day D i a b e t i c Rats to Ouabain,  and  Inotropic  and  Response o f P a p i l l a r y Muscles from C o n t r o l  70 Day D i a b e t i c Rats to Ouabain  66  68  13.  Inotropic  Response o f L e f t A t r i a to Ouabain  71:  14.  Inotropic  Response o f P a p i l l a r y Muscles to Ouabain  73  vi i  LIST OF ABBREVIATIONS  STZ  streptozotocin  ED  dose o f a g o n i s t which produces 50% o f maximum response  I  50  50  dose o f a n t a g o n i s t which i n h i b i t s 50% o f maximum response  (+)dP/dt  the maximum r a t e o f p r e s s u r e  -dP/dt  the maximum r a t e o f p r e s s u r e d e c l i n e  CK  Chenoweth K o e l l e  LVEDV  l e f t v e n t r i c u l a r end d i a s t o l i c volumes  cAMP  c y c l i c adenosine mono phosphate  cGMP  c y c l i c guanosine mono phosphate  SR  sarcoplasmic reticulum  CoA  coenzyme A  RIA  radioimmunoassay  tim  t i m o l o l maleate  iso  d,l  isoproterenol  vi i i  development  ACKNOWLEDGEMENTS  I am deeply greatful  to Dr. J . H . McNeill for his  guidance  and patience throughout my course of study. My husband, Frank McCullough has provided invaluable personal support. The expert typing of Judy Wyne is greatfully  acknowledged.  The financial support of the Medical Research Council of Canada is greatly appreciated.  INTRODUCTION  I 1.  Diabetes Mellitus and Diabetic Cardiomyopathy Introduction Diabetes mellitus  is the name given to a group of disorders  characterized by an excess of glucose in the plasma resulting from a lack of i n s u l i n or a lack of i n s u l i n a c t i v i t y . afflicted  individuals for thousands of years.  This disease has  A half century ago the  discovery of i n s u l i n by Banting and Best was heralded as a cure for diabetes (Banting and Best, 1922 and Banting et a l . , 1.922). Its therapeutic use has prevented the death of vast numbers of diabetics and_as t h e l i f e ;  of this population has increased, the r o l e - o f the Between 1897 and 1914 Jbslin  .Clinic  died  sixty in  four  percent  expectancy  c l i n i c i a n has evolved.  of • diabetics  at.  the  a • diabetic coma while cardiovascular  disease claimed only eighteen percent.  Today seventy-five  percent  die from cardiovascular complications and only one percent die in a diabetic coma (Marble,  1972).  Diabetics-  are  prone  to~  pathy, retinopathy, renal f a i l u r e , congestive heart f a i l u r e , and coronary heart disease ( West,  1978a).  In  the.  neurostroke  past.,, the  high r i s k of cardiovascular disease was attributed to the promotion of atherosclerosis study  by diabetes ( Boucher  (Kannel et a l , 1974)  diabetics  et a l . ,  1979).  The Framingham  reported that congestive heart f a i l u r e in  could not be attributed to atherosclerosis  and coronary heart  disease and that some form of cardiomyopathy must be associated with diabetes m e l l i t u s .  Ledet et a l , 1979 have concluded that the cardio-  vascular complications of diabetes cannot be f u l l y accounted for by atherosclerosis.  In l i g h t of these observations  and in an attempt  to provide improved health care, a number of studies have attempted 1  to define changes in the myocardium of d i a b e t i c s . The information available concerning cardiovascular disease in diabetics includes the results of epidemiological  studies,  reports of cardiovascular function in human diabetics and the results of studies  involving diabetic animals.  The profound effect of diabetes  on l i f e expectancy has evoked considerable interest in the epidemiology of the disease from s c i e n t i s t s ,  c l i n i c i a n s and l i f e insurance companies.  One of the most useful surveys has been the Framingham Study (Kannel et a l . , 1974).  Initiated in 1949 to explore cardiovascular disease  in 5209 men and women aged 30-62 years, this study has provided valuable information regarding the role of diabetes as an independent risk factor.  Individual case studies are o f . l i m i t e d value in terms  of defining changes in the myocardium of the diabetic population, however, some reports concerning myocardial function in groups of diabetic patients are available (Regan et a l . , 1977). The development of diabetes is influenced by a number of factors including adiposity, genetics, sex and parturition  (West,  viral  i n f e c t i o n s , d i e t , race,  1978b).  exercise,  A number of these factors  also influence the development of cardiovascular diseases. been d i f f i c u l t , i n epidemiological studies,  It has  to assess the independence  or co-dependence of these r i s k factors in the presentation of diabetes mellitus and cardiovascular disease.  The use of animal models of  diabetes has allowed control of some of these factors and has allowed researchers to observe large numbers of subjects.  Diabetes occurs  spontaneously in many species however the extreme r a r i t y has prompted scientists  to induce diabetes  in animals by various means in order  to study the disease (Mordes-and . R o s s i n i , >T981)'i -Pancreatectomy was :  2  was the f i r s t method used to induce diabetes in animals; control animals were sham operated.  The major disadvantages  of this procedure  were the trauma of major surgery and the loss of pancreatic exocrine function... Recently,selective  breeding techniques have led to the  of strains of rodents which produce a large number of diabetic • o f f s p r i n g .  Mon diabetic littermates  isolation  spontaneously  serve as controls.  The mode of inheritance is more c l e a r l y defined in some cases than in others.  Insulin l e v e l s , r e l a t i v e body weights and occurance of  ketosis are dependent on the individual model of spontaneous These animals, when a v a i l a b l e , a r e very useful  diabetes.  however they require a  large amount of space and care and therefore expense.(Mordes and Rossini, 1981). The use of 8-cytotoxic  drugs has become quite popular.  such as alloxan and streptozotocin  (STZ)  Diabetogens  s e l e c t i v e l y destroy pancreatic  8 c e l l s (Dunn et a l . , 1943 and Junad et a l . , 1967).  Both of these  drugs produce glucose intolerance in a number of animals including the rat.  Guinea pigs are not sensitive to alloxan but w i l l  STZ (Rerup, 1970).  respond to  The use of two different diabetogens should ensure  that the.observed effects on the cardiovascular system result from diabetes and not from direct toxic actions of these drugs. Alloxan and STZ produce a diabetic state which is characterized by hyperglycemia and i n s u l i n deficiency  (Rerup, 1970).  The severity  of the diabetic state is dependent on the dose of diabetogen used (Rossini et a l . , 1975 and Ganda  et a l . , 1976).  Insulin injections are sometimes given to control hyperglycemia  3  in experimental animals.  The relationships between hyperglycemia i  and the sequelae of diabetis mellitus are not c l e a r .  The severity  of the diabetic state as well as the presence or absence of exogenously administered i n s u l i n are important factors to be considered when analyzing the available l i t e r a t u r e . A relationship between the duration of diabetes and the development of heart disease has been suggested (West, 1978a-)_. One would expect that a cardiomyopathy associated with diabetes might not be evident in newly diabetic animals but might appear and worsen with the duration of the disease.  It is therefore important.when examining reports of  cardiovascular function in diabetic animals,to determine the duration of the diabetic  2.  state.  Functional and Corresponding Morphological Changes in the Diabetic Myocardium ^ 9 e  a n  et a l . (1977) observed that hearts of human diabetics were  unusually s t i f f as evidenced by an elevation  in l e f t ventricular end  d i a s t o l i c pressure,and a reduction in end d i a s t o l i c volume.  A similar  observation was made in the hearts of dogs made chronically diabetic with alloxan (Regan et a l . , 1974).  The diabetic heart also appears  to have a longer pre-ejection period, a shorter l e f t ventricular ejection time, a higher r a t i o of pre-ejection period to l e f t ventricular ejection time (Ahmed et a l . , .1975).as well as a prolonged isovolumic relaxation time (Ahmed et a l . , 1975 and Rubier et a l . , 1978). Recently, Regan et a l . (1981) compared the function of hearts of one year alloxan diabetic dogs with those of non diabetic dogs.  One half of  r  the diabetic animals were maintained throughout the study on d a i l y  4  insulin injections. studied in v i v o .  Animals were anaesthetized  and hearts were  Basal l e f t ventricular function and c o n t r a c t i l i t y  were not different in diabetics as compared to controls.  During  intraventricular infusion with s a l i n e , end d i a s t o l i c pressure reached higher levels in both diabetic groups than in control animals. Although basal l e f t ventricular end d i a s t o l i c volumes (LVEDV) were not d i f f e r e n t ,  hearts from diabetic dogs displayed smaller LVEDV in  response to the saline infusion.  This would support Regan's e a r l i e r  observation of increased stiffness in diabetic hearts (Regan et a l . , 1977).  These hearts were not hypertrophied and electron microscopy  did not reveal any abnormalities in subcellular organelles.  Hearts  from both diabetic groups displayed s i g n i f i c a n t elevations of collagen which the authors suggested might account for the diminished compliance. It was noted that control of hyperglycemia by i n s u l i n was not  sufficient  to prevent cardiovascular changes resulting from chronic diabetes mel1itus. Baandrup et a l . (1981) reported that the r e l a t i v e amount of connective tissue in hearts of poorly c o n t r o l l e d , 9 month STZ diabetic rats was s i g n i f i c a n t l y greater than in well controlled or non-diabetic rats.  The progressive nature of myocardial abnormalities is  emphasized  by the fact that Modrak (1980) could not detect any changes in collagen concentration or synthesis in the hearts of rats 3, 6, 18 or 26 weeks after the induction of STZ diabetes.  Collagen concentration did  increase in both control and diabetic hearts at 18 and 26 weeks however this was probably an age related change. Haider et a l . (1981) investigated  the effect of an atherogenic  diet (high in saturated fat and cholesterol)  5  on the myocardium of  eighteen month alloxan diabetic rhesus monkeys.  Animals were  anaesthetized and heart function was monitored i n v i v o . Intraventricular saline injections producing increases  in preload resulted in decreased  stroke work in both diabetic groups (control diet and atherogenic as compared to non-diabetic groups. in dogs (Regan et a l . ,1981)  diet)  As had been previously reported  l e f t ventricular end d i a s t o l i c pressure  was increased more in diabetics while l e f t ventricular end d i a s t o l i c volume increased to a lesser extent. the theory of increased stiffness soluble  collagen decreased,  Again these observations support  in the diabetic myocardium.  While  .insoluble-collagen increased in diabetics  and might contribute to the wall s t i f f n e s s .  Occlusive lesions were  not detected in transmural coronary arteries in any of the groups. The atherogenic diet alone did not appear to produce any great- effect on cardiac performance. Fein et a l . (1980) examined l e f t ventricular papillary muscles from streptozotocin (STZ) diabetic rats 5; 10 and 30 weeks after the induction of diabetes.  Isometric studies demonstrated that diabetic  tissues had a decreased a b i l i t y to relax; the time to one half relaxation was prolonged and the maximum rate of tension decline was decreased. There were no differences  in the passive or active  curves of diabetics and controls. peak v e l o c i t i e s tissues.  length-tension  Isotonic studies revealed that the  of shortening and relaxation were lower in diabetic  From force v e l o c i t y curves i t was evident that diabetics  displayed depressed shortening v e l o c i t i e s  over a wide range of loads.  The mechanical changes were not s i g n i f i c a n t l y altered by the duration of diabetes  (5, 10 or 30 weeks).  Vadlamudi et a l . (1982) also investigated the time course of  6  development of functional changes in diabetic rat hearts.  The  function was assessed by measuring the maximum rates of pressure increase (+,dP/dt) and decrease (-dP/dt) in response to changes in atrial  filling  pressure in isolated perfused working hearts.  Hearts  from 7 day alloxan diabetic rats did not respond d i f f e r e n t l y from those of control animals however 30, 100 and 240 days after the induction of diabetes, these hearts responded to high f i l l i n g depressed . p o s i t i v e and negative DP/dt. at high a t r i a l  filling  pressures with  Depressed cardiac performance  pressures was observed in hearts from STZ  diabetic rats 100, 180 and 360 days after treatment.  The performance  of hearts from 7 and 30 day STZ diabetic rats was not different from control.  The authors suggested that cardiac functional alterations  may appear in diabetic rats about 30 days after the induction of the disease. M i l l e r (1979) reported that isolated perfused working hearts from 3 day alloxan diabetic rats demonstrated a decreased a b i l i t y to respond to increased a t r i a l  filling  pressures.  Coronary flow was not affected  however a decrease in a o r t i c output led to a decrease in cardiac output in hearts from diabetic animals. The effect of 2, 6, 10 and 28' day i n s u l i n therapy on the mechanics of 6-10 week STZ diabetic rats was also investigated  (Fein et a l . , 1981).  Neither 2 nor 6 day therapy had any effect on the depressed cardiac muscle performance previously described (Fein et a l . , 1980), however, after 10 days there was an improvement and following 28 days of i n s u l i n therapy the mechanical changes were no longer evident.  The addition  of i n s u l i n to tissue baths did not reverse the mechanical changes in diabetic t i s s u e s .  7  Penpargkul et a l . , (1980) induced diabetes  reported that 8 weeks after STZ  isolated working rat hearts demonstrated diminished  cardiac output and stroke work at high f i l l i n g left  pressures.  Maximum  ventricular pressure and maximum a o r t i c flow rate were not as  great in diabetic hearts as in controls.  The maximum rate of pressure  decline, an index of relaxation, was depressed in hearts from diabetic animals.  In agreement with Regan et a l . (1981) and Haider et a l . (1981),  these authors observed a diminished l e f t ventricular end d i a s t o l i c volume response to increases  in pressure.  The hearts from diabetic  animals were smaller than those from controls and the increase in volume expressed per gram of heart weight was actually greater in diabetic hearts than controls.  3.  Autonomic Neuropathy in Diabetes Mellitus Autonomic neuropathy is .commonly encountered in diabetics  i t s etiology remains a puzzle.  however  Within 3 to 4 days of the induction of  diabetes with STZ Kaul and Grewal (1980) observed an increase in sympathetic a c t i v i t y reflected by an increased urinary excretion of catecholamines. from c o n t r o l s . increases  Noradrenaline content in diabetic hearts was not different Twelve day STZ diabetic rats demonstrated s i g n i f i c a n t  in both serum and ventricular noradrenaline (Paulson and Light,  1981). Senges et a l . of alloxan diabetes  (1980) reported that 100 days after the induction in rabbits various changes were evident in auto-  maticity and conduction including lower sinus r a t e .  Small coronary  arteries appea'red normal however i n t r a c e l l u l a r glycogen accumulation was increased and mitochondria appeared abnormal.  8  Savarese and  Berkowitz (1979) suggested that the bradycardia reported in diabetic animals might be due to a decrease in the number of B adrenergic receptors.  These authors reported a 24% decrease in heart rate of 2  month STZ diabetic rats accompanied by a 28% decrease in 8 receptors in ventricular t i s s u e . The authors did• • not address the question as to the effect on functioning versus .spare  8 receptors.  Foy and Lucas (1976) observed a decreased s e n s i t i v i t y proterenol  to  iso-  in hearts of 1 week alloxan and 2 week STZ diabetic r a t s .  Vadlamudi and McNeill reported similar inotropic responses to isoproterenol  in hearts from alloxan or STZ diabetic animals.  Experiments  were performed at various time points between 7 days and 6 months after the administration of diabetogen. of isoproterenol  The cardiac relaxant  effect  (-dP/dt) was depressed in hearts from diabetic animals  7 days, 30 days and 6 months after the induction of diabetes (Vadlamudi and McNeill, 1980, Vadlamudi and McNeill, 1981a and Vadlamudi and McNeill 1981b).  These observations  suggest that there may be a link  between the observed defect in relaxation (Regan et a l . , 1981) and the autonomic neuropathy. Miller  et a l , (1981) investigated  the effect of 3 to 4 day alloxan  diabetes on the response of isolated perfused rat hearts to epinephrine. Diabetic hearts displayed decreased epinephrine-mediated increases  in  cAMP and protein kinase activation however the conversion of phosphorylase b to a was increased in diabetic hearts.  Propranolol, a  8 blocker,  prevented the epinephrine-induced increase in cAMP and protein kinase a c t i v i t y in control and diabetic animals and while i t also blocked the epinephrine induced phosphorylase activation in control hearts i t to i n h i b i t this conversion in hearts from diabetic animals. 9  failed  The a agonist  phenylephrine had no effect on cAMP or protein kinase a c t i v i t y and activated phosphorylase a c t i v i t y in diabetic but not control hearts indicating the possible involvement of an  a receptor.  Phosphorylase  activation in diabetic hearts was also more sensitive to glucagon, a hormone which does not stimulate  a or  3 receptors.  Glucagon-  mediated cAMP and protein kinase activation were not different in control as compared to diabetic t i s s u e s .  The decrease in cAMP and  protein kinase activation in diabetic hearts reported in this paper is consistent with the report of a decrease in  3 receptor number.  The increased s e n s i t i v i t y of phosphorylase activation may represent an a receptor involvement or perhaps a direct modification of the phosphorylase. Inqebretsen et a l . (1981) reported that alloxan diabetic rats maintained on i n s u l i n for at least 2 weeks and withdrawn from i n s u l i n 4 days prior to study displayed no change in basal cAMP, cGMP or protein kinase or phosphorylase a c t i v i t i e s . proterenol  Diabetes depressed  iso-  induced changes in cAMP and protein kinase a c t i v i t y but had  no effect on phosphorylase activation and increased l e f t ventricular pressure.  The authors suggested that i n s u l i n alters the a b i l i t y of  the heart to accumulate cAMP and interferes with the gain of the amplification cascade  system.  Das (1973) reported that one week STZ diabetes  had no effect on  adenylate cyclase a c t i v i t y of rat hearts however i t did result in a depression of cAMP phosphodiesterase Vadlamudi and McNeill  activity.  (1982) reported that the time course of  isoproterenol mediated cAMP production was not altered by 3 or 30 day  10  alloxan or STZ induced diabetes.  Basal phosphorylase a c t i v i t y was  enhanced in both 3 and 30 day diabetic tissues. increases  Isoproterenol  induced  in phosphorylase activation were greater in hearts from  diabetic animals at both time points. Vagal involvement in diabetic autonomic neuropathy has received very l i t t l e attention however slow gastric emptying and decreased; gastric secretory responses to hyperglycemia suggest a possible (Vaisrub, 1978).  involvement  Functional studies have indicated that, under certain  conditions, the diabetic myocardium has a decreased a b i l i t y to relax (Regan et a l . , 1981, Vadlamudi and McNeill, 1981a).  Cholinergic  stimulation produces a negative inotropic response in cardiac tissue and i t has been used to investigate myocardial r e l a x a t i o n . Foy and Lucas (1976) reported a decreased s e n s i t i v i t y in hearts of 1 to 2 week alloxan and STZ diabetic r a t s . McNeill  (1981c) observed no difference  to  acetylcholine  Vadlamudi and  in the response to carbachol of  isolated perfused working hearts from 7 or 30 day alloxan or STZ diabetic rats as compared to controls. sensitivity  These authors observed a decreased  to carbachol in hearts from 100 day alloxan or STZ diabetic  rats (Vadlamudi and McNeill, 1980) STZ diabetic animals.  as well as in hearts from one year  These authors also reported an increased  sensiti-  v i t y to this drug in hearts from 6 month STZ and 8 month alloxan diabetic rats (Vadlamudi and McNeill, 1981c).  4.  Diabetes and Myocardial Metabolism Free fatty acid represents approximately 60% of the  requirement of healthy hearts (Fig 1).  11  substrate  The remaining substrate  is  Fig  1.  The Substrate Supply of the Normal Heart  Glucose, lactate and free fatty acids (FFA) are the major myocardial fuels accounting for 30%, 10% and 60%, respectively, of the oxygen uptake in the fasted, basal state.  -from  Opie,  1978.  12  SUBSTRATE SUPPLY NORMAL HEART  FIG 1  cm  13  provided by glucose (30%) and lactate  (10%)  (Opie et a l . 1971).  Diabetes has a profound effect on the metabolic state of the heart. The rate l i m i t i n g step in glucose metabolism is transport across plasma membrane.  In the absence of i n s u l i n this  reduced resulting in a  the  is d r a s t i c a l l y  decrease in i n t r a c e l l u l a r glucose and hence  a decreased contribution as a metabolic f u e l .  The r e l a t i v e c o n t r i -  bution of free fatty acids is increased and this in turn i n h i b i t s several  steps in glycolysis  including glucose transport (Neely et a l . ,  1969), glucose phosphorylation (Randle et a l . , 1966) dehydrogenase  (Kerbey et a l . , 1976).  and pyruvate  Feuvray et, a l . (1979) suggested  that the enhanced oxidation of endogenous l i p i d s in diabetic might explain their resistance transport.  to i n s u l i n stimulation of glucose  The increased fatty acid oxidation in diabetic hearts  accompanied by altered levels of metabolites. triglycerides. acetyl  hearts  (Denton and Randle, 1967),  CoA, c i t r a t e (Randle et a l . , 1966)  is  Tissue levels of  long chain acyl CoA, and acylcarnitine  et a l . , 1979) are increased in diabetic hearts.  (Feuvray  Paulson and Crass (1980)  have demonstrated an increase in t r i g l y c e r i d e s in the hearts of 12 day STZ diabetic rats which was prevented by i n s u l i n treatment.  Murthy  and Shi pp (1980) have shown a correlation between heart t r i g l y c e r i d e content and t r i g l y c e r i d e synthesis in normal and diabetic rats and have suggested that the t r i g l y c e r i d e accumulation in diabetic  hearts  is due, at least in part, to accelerated t r i g l y c e r i d e synthesis. has also been reported that t r i g l y c e r i d e s are elevated muscles of human diabetics  (Alavaikko  The i n h i b i t i o n of glycolysis  14  It  in p a p i l l a r y  et a l . , 1973).  in the diabetic myocardium may have  little  effect when the heart is performing at submaximal capacity  however the diabetic heart may be less able to withstand conditions, such as increased cardiac work and anoxia, where the heart r e l i e s more heavily on the energy produced from g l y c o l y s i s . investigated  Hearse et a l . (1975)  the a b i l i t y of isolated perfused working hearts from  6 to 9 day STZ diabetic rats to survive and recover from a 30 minute period of anoxia.  Hearts from non diabetic animals recovered very  well however hearts from diabetic animals displayed an i n i t i a l rapid recovery phase followed by a period of cardiac f a i l u r e and a second, less e f f e c t i v e ,  period of recovery.  Feuvray et a l . (1979) reported that hearts from 2 day alloxan diabetic rats responded to low levels of cardiac work in a similar manner to hearts from non diabetic rats when mechanical function was determined on isolated perfused working hearts.  Hearts from both  groups of animals recovered equally well from a mild form of whole heart ischemia however hearts from diabetic animals recovered less well  from a more severe ischemia.  acid metabolites  Tissue levels of the free  fatty  long chain acyl CoA and long chain acyl carnitine  esters were elevated  in hearts from diabetic animals.  The f a i l u r e of  hearts from diabetic animals was associated with a greater increase in acyl CoA and acyl carnitine esters than occured in hearts from nondiabetic animals.  These observations  support the suggestion that the  diabetic myocardium is able to function adequately under submaximal conditions and that i t s early f a i l u r e when exposed to severe ischemia or high work loads may result from an altered metabolic state. Ingebretson et a l . (1980),using alloxan diabetic rats maintained on i n s u l i n for at least 2 weeks and then withdrawn 4 days prior to 15  experiments, observed, " in ^isolated perfused working hearts a decrease in basal l e f t ventricular pressure development and the maximum rate of l e f t ventricular pressure development (+0P/dt) when compared to controls.  There was no difference in coronary flow or  cardiac output of hearts from diabetic animals as compared to controls. Both groups recovered equally well following a ten minute period of anoxia however when the afterload  was increased by  diabetic animals demonstrated a decreased a b i l i t y anoxia.  71%, hearts from to recover from  This difference could not be overcome by increasing the  extracellular  glucose.  The observation of high levels of plasma free fatty acids in the hearts of diabetic subjects has prompted investigators the diabetic myocardium with the ischemic myocardium.  to compare  Shug et a l .  (1975) observed an increase in the concentration of long chain acyl CoA esters and a decrease in adenine nucleotide translocase  in canine  hearts following ischemia produced by l i g a t i o n of the anterior coronary artery.  Lopaschuck et a l . (1981) reported that long chain acyl  carnitines  were increased in microsomal sarcoplasmic reticulum from 4 month alloxan and STZ diabetic r a t s .  Similar increases  in free fatty acid metabolites  were reported by Feuvray et a l . (1979), Denton and Randle (1967) and Randle et a l . (1966).  5.  Function of the Sarcoplasmic Reticulum in Diabetes Diabetic hearts observed in vivo and in v i t r o have a decreased  a b i l i t y to relax (Regan et a l . 1981, Vadlamudi and McNeill 1981a). The sarcoplasmic reticulum is important in modulating cardiac relaxation (Tada et a l .  1978).  Penpargkul et a l . (1981) reported a decreased  16  calcium uptake into sarcoplasmic reticulum from hearts of 4 to 9 week STZ diabetic r a t s .  The a c t i v i t i e s  Mg )-ATPase were also depressed. ++  of Mg -ATPase and ( C a ++  + +  +  A depression of calcium transport  was also observed by Lopaschuk et a l . (1981) in cardiac sarcoplasmic reticulum from 4 month alloxan and STZ diabetic animals.  Calcium  transport was depressed in sarcoplasmic reticulum from control and diabetic hearts by  yM concentrations of palmityl c a r n i t i n e , the most  abundant of the long chain acyl c a r n i t i n e s .  The authors suggested  that the decreased calcium uptake might result from an inhibitory influence of the long chain acyl carnitines which they reported to be elevated  in the diabetic hearts.  This hypothesis  provides a link  between the metabolic derangement of the diabetic heart and the observation of depressed relaxation.  6.  The Effect of Diabetes Mellitus on the Contractile Units of the Myocardium; The functional changes observed in the diabetic myocardium might  result from a modification of myocardial c o n t r a c t i l e elements. et a l . (1981) reported that the a c t i v i t i e s actomyosin as well as C a  + +  of C a  + +  Malhotra  ATPase from cardiac  ATPase and actin activated ATPase from  pure myosin are s i g n i f i c a n t l y depressed in rats as l i t t l e as one week after the induction of STZ diabetes. a decrease in m y o f i b r i l l a r C a  + +  Pierce and Dhalla (1981) observed  ATPase a c t i v i t y in rats eight weeks  after the induction of diabetes with STZ.  Fein et a l . (1981) reported  that i n s u l i n therapy in 6 to 10 week STZ diabetic rats caused a gradual recovery of actomyosin and myosin ATPase a c t i v i t i e s , (1980) reported a s i g n i f i c a n t depression C a  17  + +  however Dillman  ATPase from actomyosin  and myosin of 8 week STZ diabetic rats which had been maintained on i n s u l i n for the f i n a l 4 weeks.  7.  Summary Diabetes mellitus  many t i s s u e s . myocardium.  produces a change in the metabolic state of  Free fatty acid oxidation is enhanced in the diabetic Relaxation is impaired in hearts from chronically diabetic  animals and this results  in increased s t i f f n e s s .  This defect may be  due to enhanced levels of connective tissue or to a defect in calcium uptake by SR. sensitivity  The diabetic heart  also appears to have an altered  to both adrenergic and cholinergic agents possibly as a  consequence of autonomic neuropathy.  Increased workloads and periods  of sustained anoxia are tolerated less well by hearts from diabetic animals than controls.  There is some evidence that diabetes produces  a defect in the c o n t r a c t i l e machinery of diabetic  18  hearts.  II. 1.  Cardiac Glycosides and the Heart Introduction The observation that the high frequency of congestive heart  f a i l u r e among diabetics could not be explained by known r i s k led  factors  Kannel et a l . (1974) to suggest that "...diabetes is another  discrete cause of congestive heart f a i l u r e and that some form of cardiomyopathy is associated with d i a b e t e s . . . " . cardiac glycosides  Despite the use of  and potent diuretics congestive heart f a i l u r e  remains a dangerous and very often lethal  condition.  The medicinal value of cardiac glycosides since ancient times.  has been recognized  The Egyptians, Chinese and Romans u t i l i z e d  these drugs for their therapeutic as well as toxic properties-. These drugs are today used in the treatment of a t r i a l flutter,  f i b r i l l a t i o n and  paroxysmal tachycardia, sick sinus syndrome and, most importantly,  in the treatment of congestive heart f a i l u r e .  This is a common end  point for many cardiovascular disorders such as atherosclerosis  or  rheumatic myocarditis and may occur following a myocardial i n f a r c t i o n (Moe and Farah, 1975). The f a i l i n g heart has a decreased capacity to develop force during systole.  This results  in a lowering of the S t a r l i n g Curve.  For a  given cardiac output, the f a i l i n g heart must develop a greater end d i a s t o l i c pressure than a healthy heart.  The maximum cardiac output  of a f a i l i n g heart is much less than that of a normal heart. inefficient  The  f a i l i n g heart f a i l s to. empty t o t a l l y with each contraction.  For a given pressure the volume of a f a i l i n g heart is much greater than that of a healthy heart.  Despite the body's attempt to compensate  for the decrease in ejection volume by increasing heart rates the 19  cardiac output remains reduced in congestive heart f a i l u r e .  There  is an inadequate perfusion of organs (Moe and Farah, 1975). Cardiac glycosides  are of value in the treatment of heart  f a i l u r e due to t h e i r direct positive inotropic action which causes the v e n t r i c l e to develop more tension and eject more f l u i d vs. a given after load.  The increase in stroke volume and hence, cardiac  output allows the heart to empty more adequately and leads to a decrease in end s y s t o l i c and end d i a s t o l i c volumes (Moe and Farah, 1975).  2.  Role of (Ma + K )-ATPase in the Positive Inotropic Action of Cardiac Glycosides +  +  The effect of cardiac glycosides  observed in vivo results  actions of these drugs on mechanical and e l e c t r i c a l  from  properties of  the myocardium as well as actions on the nerves which innervate the heart.  The use of isolated tissue preparations has enabled i n v e s t i -  gators to examine the effect of d i g i t a l i s in the absence of innervation. The sodium pump is responsible for actively transporting Na and +  K  +  against t h e i r electrochemical gradients and thereby allowing c e l l s  to maintain cytoplasmic concentrations of Na less than and K +  than those in the e x t r a c e l l u l a r f l u i d .  In 1957  +  greater  Skou described an  ATPase from crab nerve membranes which was stimulated by Na and K +  in the presence of M g  ++  +  and suggested that this enzyme could provide  the physiological mechanism for maintaining the low cytoplasmic concentrations of Ma and high cytoplasmic concentrations of K +  (Skou,  1957).  +  In the quarter century since this observation,  it  has become quite firmly established that (Na + K )-ATPase is indeed +  the sodium pump.  20  +  Cardiac glycosides (Na  +  + K )-ATPase.  are potent and specific  inhibitors of  In 1960 Skou reported that the Na plus K  +  +  +  stimulated ATPase a c t i v i t y which he had previously described, could be inhibited in a dose dependent manner by ouabain (Skou, 1960). Ouabain, and to a lesser extent other cardiac glycosides, u t i l i z e d as tools in the study of the enzyme. (1979) stated,  have been  Robinson and Flashner  "ouabain i n h i b i t s the sodium pump, and those fluxes  that ouabain i n h i b i t s are fluxes through the .'sodium-pump". The sodium  pump is generally thought tp expend energy for  the movement of K in and Na out of c e l l s . +  Glynn et a l . (1975)  +  have reported that,  in erythrocytes, the pump can exist in four  transport modes a l l of which are sensitive to ouabain. i) coupled N a / K +  +  exchange,  exchange and iv) K / K +  Na  +  +  ii)  uncoupled Na efflux, +  exchange.  iii)  Na /Na +  +  In terms of maintaining desired  +  +  +  and K gradients, the coupled Na /K  important.  These are  exchange mode is the most  The reported stoichiometry varies  with enzyme source,  however, the erythrocyte pump appears to operate with 3Na /2K /ATP. +  (Sen and Post, 1964).  The resulting net outward movement of  change contributes to the resting membrane potential  of i n h i b i t i n g (Na + K )-ATPase. +  +  positive  of the c e l l .  There is l i t t l e doubt that ouabain and other cardiac are capable  +  glycosides  There remains, however,  some question as to the relationship between this i n h i b i t i o n and the positive inotropic effect of cardiac glycosides tissues.  on myocardial  It has been reported that very low concentrations  cardiac glycosides may stimulate the sodium pump and s t i l l  of  produce  a positive inotropic effect (Godfraind and Ghysel-Burton, 1977 and Ghysel-Burton and Godfraind, 1979). This dilemma w i l l be addressed 21  in a l a t e r section.  The majority of evidence to date links the  inotropic effect to an increase in exchangeable calcium which has been observed following exposure of cardiac tissues to  glycosides.  In 1964 Langer proposed the existance of a l i n k between i n t r a c e l l u l a r Na  concentration and C a  +  that such a N a / C a +  + +  in response to cardiac glycoside i n h i b i t i o n  (Na + K )-ATPase, there is an increase in i n t r a c e l l u l a r N a +  +  to an increase in N a / C a +  intracellular C a Ca  Considerable evidence suggests  exchange mechanism does exist in cardiac t i s s u e .  + +  It has been proposed that, of  influx.  + +  + +  + +  +  leading  exchange and therefore an increase in  concentration (Langer and Serena, 1970).  This  may be available to interact with the c o n t r a c t i l e elements  and produce the inotropic event.  This hypothesis  has become very  popular however a d e f i n i t i v e proof of i t s v a l i d i t y has eluded  investi-  gators. The inotropic response to cardiac glycosides onset.  has a very slow  A possible explanation could be that (Na + K )-ATPase is +  a c a r r i e r of cardiac glycosides, f l u i d to i n t r a c e l l u l a r s i t e s .  +  moving the drugs from the e x t r a c e l l u l a r  A recent paper by Yamamoto et a l . (1981)  provides strong evidence against this hypothesis.  They reported  that the a f f i n i t y of ( N a + K )-ATPase for ouabain was almost the +  +  same when prepared from guinea pig l e f t a t r i a , right v e n t r i c l e or p a p i l l a r y muscles. of  The number of glycoside binding sites per unit  protein and the (Na + K )-ATPase a c t i v i t y were greater in prepara+  +  tions from right v e n t r i c l e or p a p i l l a r y muscles than those from l e f t  86 a t r i a homogenates. cells,  Ouabain-sensitive  Rb uptake into  intact  an index of sodium pump a c t i v i t y , was greater in p a p i l l a r y  muscle preparations than in l e f t a t r i a l  preparations.  The rate of  onset of the positive inotropic response to ouabain was not different  22  in right v e n t r i c l e and p a p i l l a r y muscles compared to l e f t a t r i a despite the higher (Na + K )-ATPase concentration and greater +  +  capacity for active transport of  monovalent cations  preparations as compared to l e f t a t r i a . transporter of the glycosides,  in these two  If the enzyme was  the  one would predict that the onset of  ouabain-induced inotropy would be more rapid in tissues displaying higher concentrations of enzyme.  The magnitude of the  should be related to the concentration of enzyme.  response  The inotropic  response to ouabain was greater in l e f t a t r i a and right v e n t r i c l e as compared to p a p i l l a r y muscles while the enzyme concentration was greater in both right v e n t r i c l e and p a p i l l a r y muscles than in l e f t atria. If (Na + K )-ATPase is the mediator of the positive inotropic +  +  effect of cardiac glycosides  in cardiac t i s s u e s , one would expect  to find the enzyme in such tissues and indeed there is l i t t l e doubt regarding i t s  presence.  Furthermore,  specifically  on  the enzyme.  employed  photo  or  near  l a b e l l i n g techniques  binding s i t e on the  a  these drugs In 1974  Ruoho  to demonstrate  subunit of (Ma + K )-ATPase. +  a f f i n i t y l a b e l l i n g and other techniques  should bind  +  investigators  the  and Kyle digitalis  Using photoare  now  attempting to define the molecular characteristics of the binding sites for cardiac glycosides  on (Na + K )-ATPase. +  +  If enzyme i n h i b i t i o n is the cause of the inotropic response, enzyme i n h i b i t i o n should be detected before, or at least , at the same time as,the inotropic response is observed. data, one is faced with technical  problems of enzyme assays and  data which have not been independently confirmed. 23  Examining the  Ok'ita and co-workers  reported no change in enzyme a c t i v i t y following 3 hour incubation with a concentration of cardiac glycoside which produced a 50-80% increase in tension.(Roth-Schechter  et a l . , 1970).  In a l a t e r paper Okita's  group was able to demonstrate enzyme i n h i b i t i o n which persisted following a washout of drug which was accompanied by a loss of the inotropic effect (Okita et a l . , 1973).  The observations of Bentfeld  et a l . (1977) support these observations however enzyme i n h i b i t i o n was reversible at stimulation frequencies stimulation frequencies  less than 4 Hz.  At greater  the i n h i b i t i o n became i r r e v e r s i b l e .  It  is  possible that the continued i n h i b i t i o n of the enzyme could result from hypoxia due to cardiac glycoside induced increases of contraction and,vasoconstriction.  in force  Akera et a l . (1973) suggested  that f a i l u r e to detect enzyme i n h i b i t i o n at the time of the maximum inotropic action could be attributed to the observation that the half l i f e of dissociation of the drug-enzyme complex is close to the h a l f l i f e of the offset of the inotropic response. As stated e a r l i e r , there is l i t t l e doubt that (Na + K )-ATPase +  is the sodium pump.  +  If enzyme i n h i b i t i o n is responsible for the  inotropic response to cardiac glycosides, accompanied by Na pump i n h i b i t i o n .  this response should be  Ouabain sensitive  Rb  uptake  into c e l l s preloaded with Na, is reduced when guinea pig ventricular s l i c e s are prepared during the inotropic response that  follows  cardiac glycoside administration (Akera et a l . , 1975).  As the  inotropic effect increases,  so does the pump  and Smith (1978), using biopsy techniques,  inhibition.  reported  a  Hougan  decrease in  sodium pump a c t i v i t y concomittant with an increase in maximum +DP/dt.  24  If cardiac glycosides  produce t h e i r positive inotropic effect  as a result of (Na + K )-ATPase i n h i b i t i o n i t +  +  would follow that  any drug which i n h i b i t s the enzyme should also evoke a positive inotropic response.  A number of agents have been shown to produce  such an effect in cardiac tissues at concentrations that i n h i b i t (Na  +  + K )-ATPase. +  benzoate,  These include N-ethylmaleimide, p-chloromercuri-  prednisolone, 3,20-bisguanylhydrazone,  ethacrynic a c i d ,  f l u o r i d e , doxorubicin, sanguinarine, cassain, Rb  and Tl  (Akera and  Brody, 1978). Akera et a l . (1975) demonstrated that, over a period of 60 minutes, the amount of digitoxin bound to the enzyme correlated very well with the change in c o n t r a c t i l e force.  Digitoxin binding  increased with c o n t r a c t i l e force over a 20 minute period of drug exposure and then declined with c o n t r a c t i l e force over a 40 minute washout period.  This experiment was carried out on Langendorff  guinea pig hearts. Rhee et a l .  (1976) reported that (Na + K )-ATPase a c t i v i t y +  could not be s i g n i f i c a n t l y  reduced  +  by concentrations of ouabain  which produced an inotropic effect and only by concentrations which produced a toxic effect.  The authors suggested that this supports a  dissociation between the inotropic effect and enzyme i n h i b i t i o n . It should be noted that the enzyme a c t i v i t y was s l i g h t l y reduced by the lower concentrations of ouabain.  The authors used very small  groups (n = 4 to 6) and actually observed a depression of (Na + K ) +  ATPase a c t i v i t y * a l b e i t not s t a t i s t i c a l l y with the inotropic a c t i v i t y .  25  significant,  associated  +  Recent work from Godfraind's lab has suggested that i n h i b i t i o n of the sodium pump may not be the sole determinant in the positive inotropic effect of ouabain in guinea pig hearts.  Two specific  binding sites for ouabain have been reported (Godfraind et a l . , 1980). Low doses of some glycosides, (those with an unsaturated lactone at the C17 position)  stimulated the sodium pump and produced a  positive inotropic response in guinea pig a t r i a .  When the  concentration  of K in the buffer was changed there was a change in the ouabain +  ED50  for pump  i n h i b i t i o n ' but not for the inotropic effect  Burton and Godfraind, 1979).  (Ghysel-  Godfraind and Ghysel-Burton (1980)  also plotted the positive inotropic effect vs. pump i n h i b i t i o n  (as  42 + measured by ouabain sensitive regression  K uptake).  They reported  identical  lines using various low K solutions and in the presence of +  ymolar concentrations  of dihydroouabain, a cardiac glycoside which  does not contain an unsaturated lactone r i n g .  The regression  line  for ouabain was much steeper suggesting that there may be an additional factor contributing to the inotropic response.  3.  Low Dose Effects of Ouabain.- Possible Biphasic Inotropic Response in Cardiac Tissues  There have, ouabain.  recently,  Hougen and Smith  been reports of biphasic responses to (1980)  reported that nmolar doses of oc  ouabain could stimulate ouabain sensitive pig  left  atria  Rb uptake in guinea  however this could be blocked by 10  or pretreatment with reserpine.  The  26  authors  M propranolol  suggested  that  the  stimulation of the sodium pump caused by low concentrations  of  cardiac glycosides could be mediated by the release of endogenous catecholamines. a t r i a l and cats  displayed no inotropic  inotropic  papillary  a monophasic however  displayed  a  right biphasic  response represented  from  rabbits and  low concentrations  were unable to  guinea  cat  response  ventricular response 20-40%  pig,  in  left atria  from  to this  drug.  of the total  ventricular  or r a b b i t .  strips  of  detect a  in l e f t a t r i a - or right  inotropic  response was not altered by  reported that  from guinea pigs,  (1981)  effect  muscles  (1982)  response to  Schwartz et a l .  biphasic  rats  Grupp et a l .  ventricular tissues  ouabain.  evoked  Recently  this  Ouabain from  species The low  dose  inotropic response.  8 blockade.  The  The authors suggested  that ouabain has two binding sites and that the binding to the high a f f i n i t y s i t e leads to a direct increase  in i n t r a c e l l u l a r calcium  while binding to the low a f f i n i t y s i t e requires i n h i b i t i o n of (Na + K )-ATPase and an increase an increase  in i n t r a c e l l u l a r Na  in i n t r a c e l l u l a r C a . + +  which then produces  In both mechanisms the  in i n t r a c e l l u l a r calcium leads to contraction.  +  increase  The authors also  speculate that both of these mechanisms may be present in a l l species however the a f f i n i t y of the two receptors  for cardiac glycosides is  so similar that they cannot be distinguished.  Erdmann  et al .  (1981) reported a single ouabain binding s i t e in human, cat,  calf  and dog cardiac tissues and two binding sites in guinea pig and rat hearts.  They reported that  86  Rb uptake and (Na  27  +  + + K )-ATPase  were inhibited only when ouabain was present in  sufficient  concentrations  Erdmann et a l .  to occupy the low a f f i n i t y s i t e s .  (1980) attempted to correlate [ H] ouabain binding on isolated cardiac c e l l membranes and intact contracting tissue to ouabain induced i n h i b i t i o n of (Na ouabain induced positive out on rat hearts.  +  + 86 + K )-ATPase and Rb uptake and to  inotropy.  These experiments were carried  They reported two ouabain binding sites in  c e l l membrane-preparations and only one in intact ventricular tissue.  The high a f f i n i t y low capacity s i t e in membranes has a  KD (1.05  x 10~^M) very similar to that of the s i n g l e . s i t e in ventric-  ular tissue (3 x 10~^M).'Half maximal inotropic effect occured at 3 x 10~^M ouabain.  These authors demonstrated a maximum inotropic  effect of ouabain at 10  M while other authors  Godfraind, 1979 and Ku et a l . , 1976)  (Ghysel-Burton and  reported a maximum at ouabain  -4 concentrations  of 10 "M.  This discrepancy is very important in  l i g h t of the very narrow dose range of the ouabain dose response curve.  Erdmann et a l .  (1980) suggested that the low a f f i n i t y ,  capacity binding s i t e (KD' = 2.8 x 10  high  M) observed in membrane  tissues may be an a r t i f a c t or may represent sites unrelated to the positive  inotropic effect of cardiac  glycosides.  Grupp, Grupp and Schwartz (1981) - recently reported that a monophasic inotropic response was evoked by ouabain in rat l e f t a t r i a however a biphasic response  28  to this  drug could be observed  in rat ventricular s t r i p s . of 0.5  ED50  The low dose response had an  yM and represented about 30% of the total ouabain response.  The response e l i c i t e d by higher doses of ouabain had an  ED50 of  35 yM and represented the remaining 70% of the response.  ED50 was 16 yM ouabain. histamine  Reserpinization,  a and  The "overall"  8 blockade and  blockade had no effect on either response.  The low  dose response was abolished when tissues were subjected to a ouabain dose response curve, washed for 60 to 120 minutes and another dose response curve was performed. due to desensitization.  The authors explained that this was  ED50 for  The authors also noted that the  the high dose effect was very close to the reported value for 150 of  (Na + K )ATPase and therefore the inotropy observed following +  +  administration of high doses the enzyme.  might result from i n h i b i t i o n of  The low dose inotropic effect occurs when ouabain is  bound to the enzyme but the enzyme is not i n h i b i t e d . development did not f a l l  The tension  to predrug levels before the  response curve was performed.  It is possible that  second dose  60-120 minute washing  was not s u f f i c i e n t to remove ouabain from the high a f f i n i t y s i t e s . If these sites were f u l l y occupied, one would not expect to observe an inotropic response to low concentrations of ouabain. and Lindenmayer  Wellsmith  (1980) reported two conformations of (Na + K )-ATPase +  +  in canine sarcolemma. Ouabain w a s b o u n d - t o both .enzyme conformations, however only one conformation was involved in the production of the inotropic  response.  The action of low doses of cardiac glycosides  is poorly understood.  At present there is no clear l i n e through the c o n f l i c t i n g reports.  29  Further evidence is needed to define the inotropic response and to determine the link with sodium pump i n h i b i t i o n .  4.  Low S e n s i t i v i t y of Rat Hearts to Cardiac Glycosides The ubiquitous nature of (Ma + K )-ATPase has allowed +  investigators  to examine  number of species.  +  the action of cardiac glycosides  The myocardium of rats  is,notoriously  requiring very high concentrations of glycosides (Repke et a l . , 1965).  Investigators  in a insensitive,  to e l i c i t a response  have attempted to explain the  r e l a t i v e i n s e n s i t i v i t y of the rat myocardium. An increase in stimulation frequency causes an increase in the force of myocardial contraction in several species including guinea pig and rabbit (Kruta, 1937 and Katzung et a l . , 1957).  A similar change results  in a decrease in the force of contraction in rat hearts (Benforando, 1958)..  Blesa et a l . , (1970) and Langer (1970) proposed that the .  mechanism responsible for this difference might also be responsible for the low s e n s i t i v i t y of the rat heart to cardiac glycosides.  McCans  et a l . (1974) changed the positive staircase effect in the rabbit heart to a negative staircase effect by addition of the calcium channel blocker, verapamil.  This treatment did not decrease  the  s e n s i t i v i t y of the rabbit heart to ouabain indicating that the absence of the Rowditch phenomenon was not s u f f i c i e n t sensitivity  for loss of  to ouabain.  In 1969 Allen and Schwartz reported differences  in the binding  characteristics of ouabain to (Na + K )-ATPase preparation from +  +  3 r a t , dog and beef sources. parations in a 1:1  ratio  H glycoside bound to dog and beef prewhile rat demonstrated a 2:1 to 3:1 r a t i o . 30  The ouabain-enzyme complex from dog and beef was time and temperature sensitive while that from rat was not.  Enzyme-drug complexes from  rat tissues were disrupted by resuspension while those from dog and beef were not, indicating that the drug bound more loosely to (Na  +  + K )-ATPase isolated from rat tissues. +  The authors suggested  that, in the r a t , "the complex formed between the drug and one possible receptor is unstable and the drug probably binds to some sites unrelated to enzyme i n h i b i t i o n " . Tobin and Brody (1972) confirmed that the enzyme-ouabain complex was much less stable with rat (Ma + K )-ATPase than with +  other species.  +  Tobin et a l . (1972) suggested that species  differences  in s e n s i t i v i t y to cardiac glycosides were due to differences dissociation constants for the drug-enzyme complex.  in the  They examined  enzymes from guinea p i g , dog and cat but not rat as they were unable to demonstrate reproducible s p e c i f i c binding. If there were no difference in association rate constants and an increase in dissociation constant of enzyme-ouabain complexes in rats with respect to more sensitive species,  one would expect that a steady state level of complex  would be reached at an e a r l i e r time i n . r a t s .  A higher concentration  of ouabain would be required to reach the same steady state level as in more sensitive  species.,  Ku et a l . (1976) demonstrated that  the positive inotropic effect of various concentrations of ouabain reached a plateau in less than 10 minutes in rat a t r i a while tension continued to increase 30 minutes after exposure of guinea pig a t r i a . This observation strengthens the argument that the differences s e n s i t i v i t y to cardiac glycosides result from differences rates. 31  in  in dissociation  In 1979 Akera et a l .  compared ouabain with compounds with  altered lactone or steroid configurations inotropic e f f e c t ,  positive  time-response relationship and enzyme i n h i b i t i o n  in guinea pigs and r a t s . sensitivity  in terms of  The authors concluded that the low  of rat hearts to cardiac glycosides  results  from the  absence of a l i p i d barrier which, in more sensitive species, stabilizes results  the drug-receptor complex.  The absence of this  barrier  in the increased dissociation constant of cardiac glycoside  receptor in cardiac tissues of the r a t .  5.  Insulin and Diabetes and the (Na + K )-ATPase Mediated Ouabain Response +  Our i n i t i a l interest  +  in the response of the diabetic heart to  ouabain stemmed from the observation of the increased r i s k of congestive heart f a i l u r e in d i a b e t i c s „ ( K a n n e l glycosides  et a l . , 1974).  Cardiac  are very valuable in the treatment of this disease and i t  was f e l t that their response might be a l t e r e d .  Several other observa-  tions suggested to us that diabetes might a l t e r the response to these drugs. Diabetes produces alterations  in basement membranes of many  tissues (Friedenwald, 1950, Bergstrand and Bucht, 1957, and Siperstein et a l . , 1968).  Alterations in the environment near the (Na + K )-ATPase +  +  could a l t e r the interaction between enzyme and cardiac glycoside. Baily and Dresel (1971) reported that the inotropic response of l e f t a t r i a from 3 day alloxan diabetic rabbits appeared i n i t i a l l y similar to that of control atria, however, the response was not maintained unless i n s u l i n was added to the bathing medium. 32  The authors  concluded that sugar transport and metabolism were necessary maintenance of the positive inotropic response to cardiac Resh  et a l .  for  glycosides.  (1980) reported that i n s u l i n stimulated (Na + K ) +  86 ATPase dependent  +  + Rb  uptake in rat adipocytes.  These observations  suggest that the positive inotropic effect of cardiac glycosides may be dependent on high basal  (Na + K )-ATPase a c t i v i t y such as that +  +  produced by i n s u l i n stimulation. Onji and Liu (1980) reported that there was no difference  in  the number of ouabain binding sites in myocytes obtained from control and 5 to 8 day alloxan diabetic dogs.  The authors d i d , however,  observe a decrease in the a f f i n i t y for K in enzymes from diabetic +  animals.  The effect would be amplified by the accompanied decrease  in c e l l u l a r K content of diabetic t i s s u e s . +  Ku (1980)  reported a decrease in sodium pump a c t i v i t y in 5  week STZ diabetic r a t s .  The same laboratory observed a decrease in  the maximum inotropic response to ouabain of isolated l e f t a t r i a from these diabetic r a t s . ( S e l l e r s  and Ku, 1981).  As mentioned previously, long chain acyl carnitine levels may be increased in the myocardium of diabetic animals (Shug et a l . , 19-75). Adams et a l .  (1979)  reported that increasing concentrations  of  palmityl carnitine w i l l enhance and then i n h i b i t the binding of ouabain to (Na + K )-ATPase while a l l concentrations of palmityl +  +  carnitine w i l l i n h i b i t (Na  +  +  + K )-ATPase a c t i v i t y .  It appears that diabetes could interfere with the inotropic response to ouabain.  It is possible that this could appear in early  diabetes or i t could appear as a result of progressive changes in the diabetic heart some of which  has  33  already been discussed.  III. 1.  Summary of Experimental Aims  In l i g h t of the growing evidence for a s p e c i f i c  cardiomyopathy  associated with diabetes, i t was of interest to us to examine the response of cardiac tissues from diabetic animals to the cardiac glycoside ouabain  at: various times after the induction of diabetes.  We were interested in the q u a l i t a t i v e nature of any change as well as in defining the time point at which such a change f i r s t 2.  The observation  appeared.  of a biphasic response to ouabain in p a p i l l a r y  muscles led us to investigate more c l o s e l y , the e f f e c t of low concentrations of cardiac glycosides on the inotropic state of cardiac tissues. 3.  Autonomic dysfunction  i s common among diabetics.  terenol, we hoped to define a time point at which this became evident.  Using isoprodysfunction  We wished to compare this time point with that of  the appearance of changes in the response to cardiac glycosides.  34  MATERIALS AND METHODS  1.  Indugtfo^  Female Wistar rats of 175-250 g were made diabetic by a single dose of alloxan, 40-65 m g k g , or streptozotocin -1  (STZ), 50-60 rngkg" . The  drugs were prepared in 0.1 M c i t r a t e buffer pH 4.5. received an injection of buffer. to ether fumes inside a bell j a r .  1  Control  animals  Animals were anaesthetized by exposure The t a i l of each animal was dipped in  hot water ( r 6 0 ° ) for approximately 30 seconds then wiped b r i s k l y with a Kim Wipe soaked with ethanol. veins f a c i l i t a t i n g  This procedure dilated the l a t e r a l  tail  the intravenous administration of diabetogen or vehicle.  The solutions were administered as rapidly as possible using a 1 ml syringe with a 25 gauge needle. p  Animals were tested for glycosuria using L i l l y Tes-Tape . of 4+  A reading  corresponding to a urine glucose concentration of 2% or more,  was considered evidence of diabetes.  2.  Maintenance of Animals Animals were housed in wire cages containing 6 to 8 animals.  were provided with food (Purina rat chow) and water ad l i b i t u m .  They Approximately  18 hours prior to s a c r i f i c e food was withdrawn. Animals were stunned by a blow to the head and k i l l e d by cervical dislocation.  Blood was collected and the serum was separated by c e n t r i -  fugation and stored at -40 for l a t e r a n a l y s i s . o f glucose and i n s u l i n . Hearts were quickly removed and placed in Chenoweth-Koelle (CK) buffer  35  pH 7.4 gased with 95% 02/5% C0 at room-temperature. 2  in mM: NaCl 120; KCI 5.6; C a C l  2  2.2; MgCl  2  The buffer contained  2.1; glucose 10.0; NaHCOg 19.2  (Chenoweth and Koelle, 1946).  3.  Preparation of Isolated Tissues  Left and right a t r i a were separated from ventricular t i s s u e . muscles were excised from the l e f t v e n t r i c l e . discarded.  Papillary  Branched p a p i l l a r i e s were  Left and right a t r i a were separated and trimmed of extraneous  tissue and care was taken to not damage the sino a t r i a l  node.  A l l tissues  were suspended in 25 ml tissue baths containing CK buffer pH 7.4 at 37° and bubbled with 95% 0 /5% C 0 . 2  2  Tissues were connected at one end via one or more pins to platinum stimulating electrodes and at the other end via a Palmer c l i p and s i l k suture to a Grass force displacement transducer. coupled to a Beckman R611 Dynograph All  The transducers were  or to a Grass 79D Polygraph.  tissues were adjusted to a diastolic tension of 1.0 g.  a t r i a were allowed to beat spontaneously.  Right  Left a t r i a were equilibrated  for  15 minutes and then stimulated with 5 msec square wave pulses at  3.3  Hz.using a Grass stimulator S6C or SD9.  Papillary muscles were  immediately stimulated with similar pulses at 1 Hz. A l l tissues were equilibrated for approximately 60 minutes unless otherwise stated. Tissues were washed with warm fresh oxygenated buffer every 20 minutes except during a ouabain dose-response curve which took 2 hours to complete. The tissues could not be washed during this period. where timolol was used for  3 blockade, I O  buffer at a l l times.  36  - 7  In the experiments  M timolol was present in the  All  dose response curves were performed in a cumulative manner using  a buffer volume of 20 ml. terenol  Inotropic and chronotropic responses to isopro-  reached a maximum in less than one minute.  maximum response the subsequent dose was added.  At the time of the  The inotropic response  to ouabain developed over a period of five to eight minutes.  A period of  ten minutes was allowed between the administration of each drug dose.  4.  Preparation of Drugs _2 A 10  M stock solution of  d , l isoproterenol  HC1 at the beginning, of each set of experiments.  was prepared in 0.05 M Dilutions were made in  buffer immediately before each dose response curve. were prepared in buffer on the day of the  5.  A l l ouabain solutions  experiment.  .Analysis o f Serum Serum i n s u l i n concentrations  assay (RIA) k i t  were determined using a radioimmuno  purchased from Becton Dickinson.  Serum glucose concen-  trations were determined using an Ames Blood Analyzer k i t No. 510 (colorimetric)  6.  or a Glucose  k i t purchased from Sigma.  Statistical.Analyses The Students  group to c o n t r o l .  "t" test was used to compare a single experimental Analysis of variance was used when comparing three or  more groups and significance was determined using the Newman-Keuls t e s t . A value of p<.05 was considered to be s i g n i f i c a n t  37  in a l l  experiments.  Materials  Alloxan monohydrate, streptozotocin, isoproterenol  ouabain octahydrate and d , l  HC1 were purchased from Sigma.  provided by Merck-Frosst.  38  Timolol maleate was  generously  5  RESULTS  I.  Detection of Diabetes Fasted serum glucose and i n s u l i n levels indicated that animals were diabetic  by 7 days and remained diabetic past 70 days. level  At 7 days the serum glucose  of control animals was 70.1 ± 7.34 mg % while that of STZ diabetics  was 305.4 ± 23.58 mg %.  Serum i n s u l i n levels were 42.1 ± 5.08 yU m l  in samples from control animals and 18.50 ± 2.42 yU m l STZ diabetic animals.  - 1  - 1  in samples from  At 70 days serum glucose levels from STZ diabetic  rats were 477% of c o n t r o l l e v e l s .  II.  Response to d , l Isoproterenol  in Cardiac.Tissues taken from. Rats 7-or 70  Days after the Induction of Diabetes.  A.  Inotropic  Responses  Inotropic responses to isoproterenol were measured in l e f t p a p i l l a r y muscles and l e f t a t r i a at both time points.  In the absence of drug, there  was no s i g n i f i c a n t difference in the tension developed by control or diabetic tissues (Table I ) .  Isoproterenol  produced a dose-dependent  increase in tension in both a t r i a and p a p i l l a r y muscles.  There was no  s i g n i f i c a n t difference in the response of p a p i l l a r y muscles from control or diabetic animals at 7 or 70 days (Fig 2 and 3). of tissues from diabetic animals was consistently smaller at each dose of isoproterenol  (Fig 3).  At 70 days the  response  but not s i g n i f i c a n t l y  There was also no s i g n i f i c a n t  difference in the response of l e f t a t r i a from control or diabetic animals  39  Table  I  Inotropic Responses of Cardiac Tissues to d , l Isoproterenol 7 or 70 Days After the Induction of Diabetes.  Basal • Maximum Developed Developed Tension (g) Tension (g)  Maximum Increase in Tension (g)  Tissue.  Experimental Group  n  Time Point (days)  papillary  control  8  7  0.49±0.11  0.87±0.17  0.38±0.08  muscles  alloxan  6  7  0.9H0.19  1.38±0.26  0.47±0.10  papillary  control  8  70  0.79±0.19  1.13±0.26  0.33±0.08  muscles  STZ  13  70  0.65±0.11  .0.79+0.14  0.18±0.05  left  control  4  7  0.64±0.22  1.43±0.52  0.79±0.31  atria  STZ  4  7  0.75±0.08  1.36+0.09  0.6U0.08  alloxan  5  7  0.60±0.05  1 .30+0.14  0.66±0.12  left  control  5  70  0.72±0.20  1 .3H0.20  0.59±0.06  atria  STZ  8  70  0.77±0.09  1 .31+0.10  0.54±0.05  40  Fig  2.  Inotropic Response of P a p i l l a r y Muscles from Control and 7 Day Alloxan Diabetic Rats to d , l  Isoproterenol.  Tissues were stimulated with square wave pulses at a frequency of 1 hz and were equilibrated at 37° in oxygenated CK buffer, pH 7.4, addition.  for 1 hour prior to drug  Dose response curves were performed in a  cumulative manner. Response is expressed in terms of increase in tension (g) vs. the log of the molar concentration of isoproterenol.  Each point represents  Control  °Alloxan  A  n=8;  the mean ± SEM.  n=6.  41  RESPONSE TO ISOPROTERENOL [<) CONTROL (N=8) (x) ALLOXAN (N=6)  0  LOG CONC ISO  FIG  2  42  (M)  RESPONSE TO ISOPROTERENOL i<) CONTROL (N«8) (o) STZ (N«=13)  LOG CONC ISO FIG  3  44  (M)  Fig  3.  Inotropic Response of Left Papillary Muscles from Control and 70 Day Diabetic Rats to d , l  Isoproterenol.  Tissues were stimulated with square wave pulses at a frequency of 1 hz and were equlibrated at 37° in oxygenated CK buffer, pH 7.4,  for 1 hour prior to drug addition.  Dose  response curves were performed in a cumulative manner. Response is expressed in terms of increase in tension  (g)  vs. the log of the molar concentration of isoproterenol. Each point represents °STZ  the mean ± SEM. ^Control  n=13.  43  n=8;  to isoproterenol.at 7 or 70 days (Fig 4 and 5). a consistent  At 70 days, there was  but not s i g n i f i c a n t , reduction in the response of diabetic  tissues.  B.  Chronotropic Responses  Chronotropic responses to isoproterenol were measured in right a t r i a from control and diabetic animals 7 and 70 days after the induction of diabetes.  Seven days after the induction of diabetes the basal rate was  s i g n i f i c a n t l y reduced in a t r i a from STZ animals with respect to controls (Table I I ) .  A t r i a from alloxan diabetic animals demonstrated a reduction  in basal rate which was not s t a t i s t i c a l l y s i g n i f i c a n t .  At 70 days there  was very l i t t l e difference in the basal rate of a t r i a from control and diabetic animals.  Despite the difference in basal rates, there was no  s i g n i f i c a n t difference in the maximum rate attained or the maximum increase in rate in a t r i a from control and 7 day diabetic rats (Fig 6 a/b, Table I I ) . Seventy days after the induction of diabetes a t r i a from diabetic rats demonstrated smaller responses to- isoproterenol than did a t r i a from control animals.  Although this decrease was consistent  range of the dose response curve, the difference was not significant  (Fig 7).  45  throughout the statistically  Fig  4.  Inotropic Response of Left A t r i a l Tissues from Control and 7 Day Diabetic Rats to d,l  Isoproterenol.  Tissues were stimulated with square wave pulses at a frequency of 3.3 hz and e q u i l i b r a t e d , at 3 7 ° , in oxygenated CK buffer, pH 7.4, addition.  for 1 hour prior to drug  Dose response curves were performed in a  cumulative manner.  Response is expressed in terms of increase in tension  (g)  vs. the log of the molar concentration of isoproterenol. Each point represents X  Alloxan  n=5;  °STZ  the mean ± SEM. ^Control n=4.  46  n=4;  RESPONSE TO ISOPROTERENOL i<) CONTROL (N=4) (x) ALLOXAN (N=5) (o) STZ (N*4)  .H  -10  -i—i  § 111111— i  -9  i 1111 n | —  • 111  -8 LOG CONC ISO (M)  FIG 4 47  ni|— - i — i 111 u i |  -7  Fi9  5  Inotropic Response of Left A t r i a from Control and 70 Day Diabetic Rats to d , l  Isoproterenol.  Tissues were stimulated with square wave pulses at a frequency of 3.3 hz and e q u i l i b r a t e d , at 3 7 ° , in oxygenated CK buffer, pH 7.4,  for 1 hour prior to drug addition.  Dose  response curves were performed in a cumulative manner. Response is expressed in terms of increase in tension  (g)  vs. the log of the molar concentration of isoproterenol. Each point represents °STZ  the mean ± SEM. ^Control  n=8.  48  n=5;  RESPONSE TO ISOPROTERENOL i<) CONTROL (N*=5) (o) STZ (N«=8)  49  Table  II  Chronotropic Responses of Right Atria to d , l 7 or 70 Days After the Induction of  Isoproterenol  Diabetes.  Maximum Increase in _-j Rate (beats min" )  n  Time Point (days)  control  4  7  281±20  418±11  137±25  STZ  3  7  208±7  393±11  185±11  alloxan  5  7  225±17  389+19  164±9  control  5  70  223±10  439±16  217±11  STZ  7  70  235±18  403±10  159±25  a  Basal Rate _-| (beats min".)  Maximum Rate _.j (beats min" )  Experimental Group  a  p<.05 compared to control  50  Fig 6a and 6b  Chronotropic Response of Right A t r i a from Control and 7 Hay Diabetic Rats to d , l  Isoproterenol.  Tissues were allowed to beat spontaneously and were e q u i l i b r a t e d , at 3 7 ° , in oxygenated CK buffer pH 7.4 for 1 hour prior to drug a d d i t i o n .  Dose response curves  were performed in a cumulative manner.  The chronotropic response is expressed in beats m i n ~ \ In part "a" the data are expressed in terms of absolute rate vs. the log of the molar concentration of isoproterenol. In part "b" the data are expressed in terms of increase in rate vs. the log of the molar concentration of isoproterenol. A  Control  n=4;  Each point represents the mean ± SEM. X  Alloxan  n=5;  °STZ  n=3.  51  RESPONSE TO ISOPROTERENOL (<) CONTROL (NM) (x) ALLOXAN (N=5) (o) STZ (N<=3) 440420400380360in  5  340-  CQ  £ 320 H  cr cc £ 300 ZD -J O  g280H 260 240 220 200  I—i  BR -10  FIG 6a  1111 ni)—i 11 mm  -9  -8 LOG C0NC ISO (M)  52  RESPONSE TO ISOPROTERENOL (<) CONTROL (N«=4) (x) ALLOXAN (N«=5) (o) STZ (N=3) 200-1  180H  ~  160-1  CD  .O UJ  UJ  in  120H  100-1  5 8060-  40  A  20 H  0-4  J  1  1 I I  1  -9  -10  FIG 6b  53  1 I lllll)  1  1 I I llll|  -7 LOG CONC ISO (M)  -8  1  PTTTTTIJ i  -6  I  I  Chronotropic Response of Right A t r i a from Control and 70 Day STZ Diabetic Rats to d , l  Isoproterenol.  Tissues were allowed to beat spontaneously and were e q u i l i b r a t e d , at 3 7 ° , i n oxygenated CK buffer pH 7.4 for 1 hour prior to drug addition.  Dose response  curves  were performed in a cumulative manner.  Positive chronotropic response is expressed in terms of increase in rate beats min"^)  vs. the log of the molar  concentration of isoproterenol.  Each point  the mean ± SEM.  °STZ  A  Control  n=5;  n=7.  54  represents  RESPONSE TO ISOPROTERENOL t<0 CONTROL (N»=5) (o) STZ (N*7) 220-1  200 H  c E  180H  w 160H  •*->  CO  0  Sl40H UJ  120in  1008060 40 20 i i i uiiq  0 -10 FIG  1 I I  -9  nil^  1  l  1  l  1IIIH|  -8 -7 -6 LOG CONC ISO (M)  7  55  • " • •»"!  -5  11  ' ""H  -4  Ill  Response to Ouabain in Cardiac Tissues 7 Days, 70 Days or 6 Months  After the Induction of Diabetes  A.  Effect of Timolol on Ouabain Dose Response Curves  The rat myocardium is notoriously insensitive to the effects of cardiac glycosides.  Very high concentrations of these drugs are required  to produce a positive inotropic e f f e c t .  In order to determine whether  release of endogenous catecholamines contributed to the observed inotropic e f f e c t , dose response curves to ouabain were compared in the presence and absence of the  8 blocker t i m o l o l .  Timolol was present in the buffer, during  the e q u i l i b r a t i o n period and throughout the dose response curve, at a concentration (10  7  M) which had previously been shown to block the inotropic effect of catechol-  amines.  In the presence of timolol the resting tension of l e f t a t r i a was s l i g h t l y ,  but not s i g n i f i c a n t l y , greater than in the absence o f this drug.  As shown in  Figure 8, there was no s i g n i f i c a n t difference in ouabain dose response curves performed in the presence or absence of the  B.  8 blocker.  Inotropic Responses to Ouabain in Left A t r i a and P a p i l l a r y Muscles.  In the absence of ouabain, there was no s t a t i s t i c a l l y  significant  difference in the basal developed tension of tissues from control or diabetic animals (Table I I I ) .  At the l a t e r time points (70 days and 6  months) there appeared to be a tendency for control tissues to develop s l i g h t l y more tension.  Seven days after the induction of diabetes ouabain  produced a smaller increase in tension in l e f t a t r i a from both groups  56  Fig  8  Effect of 3 Blockade on the Positive Inotropic Response to Ouabain in Left A t r i a from Rats.  Tissues were stimulated with square wave pulses at a frequency of 3.3 hz.  Prior to ouabain exposure,  tissues  were equilibrated for 1 hour at 37° in oxygenated CK buffer, pH 7.4,  containing, where indicated 10~ M t i m o l o l . 7  Dose  response curves were performed in a cumulative manner.  Response of a t r i a is expressed in terms of increase in tension (g) vs. the log of the molar concentration of ouabain. X  CK  +  Each point represents 10"  7  M  Timolol  the mean ± SEM. . C K n=7; A  n=8.  57  RESPONSE TO OUABAIN [<) (N=7) (x) CR + TIM (N=8)  LOG CONC OUABAIN (M)  FIG 8  58  Table  III  Inotropic Responses of Cardiac Tissues to Ouabain at Various Times After the Induction of Diabetes.  Tissue  Experimental Group  n  Time Point  Basal Developed Tension (g)  Maximum Developed Tension (g)  Maximum Increase Tension (g)  left  control  6  7 days  0.42±0.09  0.75±0.16  0.33±0.09  atria  STZ  6  7 days  0.78±0.11  0.98±0.11  0.18±0.02  alloxan  4  7 days  0.64±0.06  0.86±0.11  0.23±0.06  left  control  9  6 months  1.06±0.10  1.59±0.21  0.5H0.08  atria  STZ  4  6 months  0.70±0.12  0.90±0.11  0.21±0.03  a  alloxan  6  6 months  0.73±0'.15  0.95±0.17  0.22±0.03  a  papillary  control  15  7 days  0.30±0.05  0.77±0.09  0.47±0.06  muse!es  STZ  8  7 days  0.34±0.03  0.66±0.07  0.32±0.06  papillary  control  5  70 days  0.76±0.20  1 .29±0.29  0.56±0.08  muscles  STZ  12  70 days  0.53±0.10.  0.77±0.14  0.26±0.07  a = p<.05 compared to c o n t r o l .  59  a  o f d i a b e t i c a n i m a l s compared to c o n t r o l was e v i d e n t a t a l l  (Fig  9).  This  p o i n t s o f the dose response c u r v e except the two  l o w e s t d o s e s , however a t no p o i n t was the d i f f e r e n c e significant.  difference  statistically  S i x months a f t e r the i n d u c t i o n o f d i a b e t e s t h i s  was a g a i n e v i d e n t  (Fig  1 : 0 ) . Tissues  demonstrated a s m a l l e r i n c r e a s e i n  from both d i a b e t i c  pattern  groups  t e n s i o n w i t h i n c r e a s i n g doses  ouabain than d i d t i s s u e s from c o n t r o l  animals.  This depression  response was s t a t i s t i c a l l y s i g n i f i c a n t i n t i s s u e s from a l l o x a n  of  of diabetic  -5 animals a t doses o f ouabain g r e a t e r than 3 x 1 0 were o b t a i n e d i n l e f t p a p i l l a r y m u s c l e s .  M.  Similar  Seven days a f t e r  results  the  i n d u c t i o n o f d i a b e t e s p a p i l l a r y muscles from d i a b e t i c animals c o n s i s t e n t l y demonstrated a n o n - s i g n i f i c a n t d e c r e a s e i n response to ouabain r e l a t i v e to c o n t r o l s  (Fig  11).  Seventy days a f t e r d i a b e t e s was  i n d u c e d , the response o f p a p i l l a r y muscles from STZ d i a b e t i c a n i m a l s was s i g n i f i c a n t l y l e s s than t h a t o f c o n t r o l g r e a t e r than 1 0 IV.-  - 5  a n i m a l s a t doses o f ouabain  M (Fig 1 2 ) .  E f f e c t o f Time and 3 Blockade on the P o s i t i v e I n o t r o p i c E f f e c t o f Ouabain  on C a r d i a c  Tissues.  In an attempt to e x p l a i n the b i p h a s i c dose response curves demonstrated by p a p i l l a r y muscles the f o l l o w i n g experiments were out.  to ouabain carried  L e f t a t r i a and p a p i l l a r y muscles were e q u i l i b r a t e d f o r a p e r i o d  one or t h r e e hours i n the presence or absence o f 1 0 "  7  M timolol.  to ouabain a d m i n i s t r a t i o n t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n development among groups o f a t r i a l or p a p i l l a r y t i s s u e s .  60  of  Prior tension  Dose response  Fig  9  I n o t r o p i c Response o f L e f t A t r i a from C o n t r o l  and 7 Day  D i a b e t i c Rats to O u a b a i n .  T i s s u e s were s t i m u l a t e d w i t h square wave p u l s e s a t a f r e q u e n c y o f 3 . 3 hz and were e q u i l i b r a t e d , a t 3 7 ° , f o r 1 hour i n oxygenated CK b u f f e r , pH 7 . 4 .  Isoproterenol  d o s e - r e s p o n s e curves were p e r f o r m e d , t i s s u e s were washed f o r a p e r i o d o f 1 hour and ouabain dose response curves were then o b t a i n e d .  Dose response curves were performed i n  a c u m u l a t i v e manner.  Response i s expressed i n terms o f i n c r e a s e i n t e n s i o n v s . the l o g o f the molar c o n c e n t r a t i o n o f o u a b a i n . p o i n t r e p r e s e n t s the mean ± SEM. X  Alloxan  ^Control  (g)  Each  n=6; °STZ n=6;  n=4.  ' 61  RESPONSE TO OUABAIN  62  Fig  10  Inotropic Response of Left Atria from Control and 6 Month Diabetic Rats to Ouabain.  Tissues were stimulated with square wave pulses at a frequency of 3.3 hz and e q u i l i b r a t e d , at 3 7 ° , for 1 hour in oxygenated CK buffer, pH 7.4,  prior to drug addition.  Dose response  curves were performed in a cumulative manner. Response is expressed in terms of increase in developed (g) vs. the log of the molar concentration of ouabain. point represents °STZ  the mean ± SEM.  A  Control  n=4.  *p<.05 compared to c o n t r o l .  63  n=9;  X  tension Each  Alloxan n=6;  RESPONSE TO OUABAIN (<) CONTROL (N«9) (x) ALLOXAN (N«6) (o)ST2 (N»=4) .55-i .5.45.4-  2  §.35H in  »-  .3H  ».25H 5  ,2H .15.1.05-  0-  — i —  -5  1  1—r—i—i—i—i—j—  -4 LOG CONC OUABAIN  FIG  10  64  (M)  Fig  11  Inotropic Response of Papillary Muscles from Control and 7 Day Diabetic Rats to Ouabain.  Tissues were stimulated with square wave pulses at a frequency of 1 hz and were e q u i l i b r a t e d , at 3 7 ° , for 1 hour in oxygenated CK buffer, pH 7.4.  Isoproterenol  dose response curves were  then performed, tissues were washed for a period of 1 hour and ouabain dose response curves were performed in a cumulative manner. Response is expressed in terms of increase in tension (g) the log of the molar concentration of ouabain. represents  the mean ± SEM. ^Control  n=15;  65  Each point  °STZ  n=8.  vs.  RESPONSE TO OUABAIN t<) CONTROL CN«=15) (o)STZ (N=8)  66  Fig  12  Inotropic Response of Papillary Muscles from Control and 70 Day Diabetic Rats to Ouabain.  Tissues were stimulated with square wave pulses at a frequency of 1 hz and e q u i l i b r a t e d , at 3 7 ° , for 1 hour in oxygenated CK buffer, pH 7.4.  Isoproterenol  dose  response curves were performed, tissues were washed for a period of 1 hour and then ouabain dose response curves were obtained.  Dose response curves were performed in a  cumulative manner. Response is expressed in terms of increase in tension the log of the molar concentration of ouabain. represents  the mean ± SEM. ^Control  n=5;  *p<.05 compared to c o n t r o l .  67  °STZ  vs.  Each point n=12.  RESPONSE TO OUABAIN (<) CONTROL (N=5)  (o) STZ (N=12)  .6-1  0-4  -6  •  1  111  1  -5  i  i i 1111—  LOG CONC OUABAIN (M) FIG  12  68  -4  curves to ouabain were then obtained. did not reduce the positive (Fig 13).  8 blockade  As previously observed,  inotropic effect of  ouabain in l e f t a t r i a  These tissues demonstrated v i r t u a l l y no increase in tension -5  until doses of ouabain greater than 10 no s i g n i f i c a n t  difference  M were administered.  There was  in the curves obtained from tissues which were  equilibrated for 1 hour vs. those equilibrated for 3 hours. The dose response curves obtained from p a p i l l a r y muscles were very different  from those of a t r i a (Fig 14).  Administration of ouabain to  l e f t a t r i a produced a monophasic dose response curve while p a p i l l a r y muscles responded in a bibhasic manner.  There was an i n i t i a l -7  in tension when ouabain was administered in dose accounted for less  than  50%  of the total  Administration of similar concentrations  of 10"  increase  in  increase -6  to 10"  M which  tension.  of ouabain had no effect on  tension development in a t r i a l t i s s u e s . ( F i g  13). There was very l i t t l e -6 -5 change in tension when concentrations of 10 to 10 M were added. The greatest increase in tension was observed when ouabain was administered -5 -4 to p a p i l l a r y muscles in doses of 10 to 10 M. Timolol did not block the response of p a p i l l a r y muscles to any dose of ouabain.  The other variable which was examined was time and i t appeared  to play an important role in the response of the tissues.  Papillary  muscles which had been equilibrated for three hours demonstrated a s i g n i f i c a n t l y greater response to ouabain at most points throughout the dose response curve than did tissues which had been equilibrated for only one hour.  69  Fig  13  Inotropic Response of Left A t r i a to Ouabain.  Response is expressed in terms of increase in tension vs. the log of the molar concentration of ouabain. point represents the mean ± SEM.  A  (g)  Each  l hour equilibration  V  in normal buffer  n=4;  1 hour equilibration in buffer  containing 10~ M timolol 7  normal buffer  n = 4;  +  containing 10 M timolol 7  n=4;  D  3 hour equilibration in  3 hour equilibration in buffer n=4.  In each case,  cumulative  dose response curves followed the equilibration period.  70  RESPONSE TO OUABAIN (<) 1 HR CK (N=4) (x) 1 HR CK + TIM (N*4) (o) 3 HR CK (N«4) (•) 3 HR CK + TIM (N«=4)  .6H  in  S .4ui in  .3H  .2H  .H  0-4  -8  -7  -6  -5  LOG CONC OUABAIN (M)  FIG 13  71  Fig  14  Inotropic Response of Papillary Muscles to Ouabain.  Response is expressed in terms of increase in tension vs. the log of the molar concentration of ouabain. point represents the mean ± SEM. in normal buffer  n=4;  7  n=8;  containing 10" M 7  Each  l hour equilibration  1 hour equilibration in buffer  containing 10~ M timolol normal buffer  A  (g)  n=8;  D  3  hour equilibration in  3 hour equilibration in buffer  timolol.  response curves followed the  In each case, cumulative dose equilibration.  *p<.05 compared to A **p<.05 compared to A and X ***p<.05 compared to A , X and •  72  RESPONSE  t<) 1  .6n  (x) 1 (o) 3 (•) 3  HRJ HR> HR* HR/  TO  OUABAIN  CK (N=4) CK + T I M (N=8) CK (N*8) CK + T I M (N«4)  .5H  CD  .4H  z o  in  5 .3-  ui in  .2H  .H  0-1 LOG  FIG 14 73  CONC OUABAIN  (M)  DISCUSSION  I.  Inotropic Response to d , l  Isoproterenol  The data presented here show no s i g n i f i c a n t differences  in  the positive inotropic responses to isoproterenol in isolated cardiac tissues from 7 or 70 day diabetic rats as compared to controls (Fig  2-5).  A tendency toward a depression of the responses was evident in papillary muscles and to a lesser extent in l e f t a t r i a from 70 day diabetic animals (Fig 3 and 5).-  Failure' to observe a s t a t i s t i c a l l y  s i g n i f i c a n t difference may be due to the use of a r e l a t i v e l y small number of tissues or to a very small difference in the two populations. Our hypothesis would suggest that changes might become more evident at a l a t e r time point due to their progressive nature. Although we have f a i l e d to demonstrate a s t a t i s t i c a l l y icant change, the observed depression is consistent curves and is worthy of comment. agreement  signif-  throughout the  The depression reported here is in  with the observations of Foy and Lucas (1976) of a  decreased s e n s i t i v i t y to isoproterenol in hearts from diabetic r a t s . Also  in agreement are the observations of Vadlamudi and McNeill  who reported a depression of the relaxant effect of at several time points following the induction of  (1981a)  .isoproterenol  diabetes.  If the depression observed represents a real change within the population, or even i f such a change was demonstrated at a l a t e r time point, the molecular basis for such an occurance remains unknown. Savarese and Berkowitz (1979) reported a  28% decrease in the number of  8 adrenoreceptors in cardiac tissue of 2 month STZ diabetic r a t s .  74  In 1956 Stephenson introduced the concept of spare receptors.  The  small decrease in receptor number reported by Savarese and Berkowitz would not be expected to a l t e r the magnitude of the 8 adrenoreceptor mediated response.  A second s i t e at which the response to isoproterenol could be modulated is the cAMP, protein kinase cascade system.  Ingebretson  et a l . (1981) reported a depression in isoproterenol induced changes in cAMP and protein, kinase and no change in and  hearts  from  Vadlamudi  diabetic and McNeill  activation in hearts discrepancy  in  from  in  animals while (1982)  Miller  reported  diabetic  phosphorylase  rats.  et a l . , (1981)  enhanced  evident.  notable difference in the studies is that Ingebretson  sacrifice.  phosphorylase  The reason for the  these results i s . n o t immediately  administered i n s u l i n to their animals up  until  The most  et a l . ,(1981)  4 days  prior to  Regarding the p o s s i b i l i t y of enhanced phosphorylase  a c t i v a t i o n , the role of calcium in this condition has not defined.  activation  been f u l l y  It is possible that calcium is the mediator of the enhanced  activation.  II.  Chronotropic Response to d , l  Isoproterenol  The chronotropic response to isoproterenol p a r a l l e l s the inotropic one.  Seven days after the induction of diabetes,  there was no difference  in the response of right a t r i a from diabetic animals as compared controls (Fig  6).  Seventy days after  the  induction of diabetes, a  non s i g n i f i c a n t depression was evident throughout the  75  to  dose  response  curve (Fig 7).  As noted above, f a i l u r e to demonstrate a s t a t i s t i c a l l y  s i g n i f i c a n t difference may be due to the small sample size or to a very small difference within the population. If the observed trend represents a real change one would expect that other responses, mediated by the same receptor and post receptor events, would be altered in a similar manner.  As reported here,  seventy days after the induction of diabetes p a p i l l a r y muscles and, to a lesser extent, l e f t a t r i a demonstrated s l i g h t l y depressed inotropic responses to isoproterenol and right a t r i a display s l i g h t l y depressed chronotropic responses to this drug. The molecular basis for a possible depression of the chronotropic response is not f u l l y understood however the p o s s i b i l i t i e s of an a l t e r a t i o n at the £> receptor level or at post receptor events are discussed in the section immediately preceeding this ( I .  Inotropic  Responses to d , l Isoproterenol).  III.  Response to Ouabain  Papillary muscles appeared to have a biphasic response to ouabain (Fig 11 and 12).  —6 Low doses (<10~ M) evoked a small increase in tension  however the principal  inotropic event occured when ouabain was administered  in concentrations of 10  -5  to 10  -4  M.  Left a t r i a responded in a monophasic -5 -4 manner to ouabain concentrations of 10 to 10 M (Fig 9 and 10). The observation of a nonsignificant depression of ouabain responses in 7 day diabetic tissues (Fig 9 and 11) is consistent with our hypothesis of the time dependence of the cardiomyopathy which develops with diabetes. It is interesting that at this time point, no trend was evident regarding the inotropic response to isoproterenol (Fig 2 and 4). 76  The depression of the response to ouabain was more evident at l a t e r time points.  A s t a t i s t i c a l l y s i g n i f i c a n t difference was  observed in papillary muscles from 70 day diabetic rats (Fig 12). Although the low-dose response was i n h i b i t e d , the depression was small and not s t a t i s t i c a l l y s i g n i f i c a n t . was on the high-dose response.  The major effect of ouabain  In l e f t a t r i a from 6 month diabetic  animals the maximum inotropic response to ouabain was also inhibited (F.ig 10).  These observations are in agreement with those of Sellers  and Ku (1981) who reported a decrease in the maximum inotropic response to ouabain of l e f t a t r i a from diabetic r a t s . The data presented here provide very l i t t l e  insight into the  molecular mechanism responsible for this depression however the major influence of diabetes appears to be on the high-dose effect of ouabain in papillary muscles and the response in l e f t  atria.  Although there is some confusion in the l i t e r a t u r e regarding the nature of the low-dose e f f e c t ,  i t appears that the  high-dose-effect  in p a p i l l a r y muscles and the response of l e f t a t r i a are mediated through i n h i b i t i o n of (Na + K )-ATPase (Schwartz et a l . , 1981). It +  +  appears that diabetes alters the (Na + K )-ATPase mediated positive +  inotropic effect of cardiac glycosides.  +  Ku (1980) reported a decrease  in sodium pump a c t i v i t y in hearts of 5 week diabetic r a t s .  The  levels of long chain acyl carnitines are elevated in the hearts of diabetic animals (Shug et a l . 1975), and palmityl c a r n i t i n e , the most abundant member of this group has been shown to i n h i b i t (Na + K ) +  ATPase.  +  Both of these observations suggest that a decrease in (Na + +  K )-ATPase a c t i v i t y may occur in the diabetic heart. +  Ouabain would  then have less enzyme units available for i n h i b i t i o n in the diabetic 77  heart as compared to control and would have a lesser effect on the diabetic heart.  The data presented here support such a theory:  tissues from diabetic animals displayed smaller inotropic responses to ouabain than did tissues from control animals. There has been very l i t t l e + (Na  investigation  into the a c t i v i t y of  + + K )-ATPase in the diabetic heart however Onji and Liu (1980)  reported that there was no difference in the number of ouabain binding sites in hearts of 5 to 8 day alloxan diabetic dogs.  It would be  interesting to repeat this experiment at a l a t e r time point following the induction of diabetes.  It would also be of interest to measure  the a f f i n i t y of ouabain for the binding sites in diabetic and control animals. Lindemeyer and Well smith (1980) reported two conformations of (Na  +  + K )-ATPase. +  Ouabain was bound to both enzyme conformations  however only one conformation was involved in the production of the inotropic response.  It would be interesting to investigate the  influence of diabetes on the conformation of (Na + K )-ATPase. +  +  Although the majority of evidence suggests that i n h i b i t i o n of the sodium pump is responsible for at least the major component of the cardiac glycoside induced positive  inotropic response,  the  p o s s i b i l i t y remains that these two events are not causally linked and that the effect of diabetes is at a s i t e other than (Na + K ) +  +  ATPase.  IV.  Effect of Time and.8 Blockade on the Ouabain Dose Response Curve The l i t e r a t u r e provides c o n f l i c t i n g evidence regarding the  78  inotropic response of cardiac tissues to low concentrations of cardiac glycosides.  The data presented in this study indicate that a possible  reason for the controversy is that ouabain produces a monophasic response in l e f t a t r i a and a biphasic response in p a p i l l a r y muscles from rats (Fig 13 and 14).  These observations are in agreement with  those of Schwarts et a l . (1981) who reported a monophasic response in rat l e f t a t r i a and a biphasic response in right ventricular strips with a low-dose ( E D  of 3 x 10" N!) and a high-dose ( E D 7  5Q  5 Q  of 3.5 x 10"  Although we were unable to calculate accurate ED^Q values due to the steepness of the curves i t is evident in Fig 14 that the response which we observed in papillary muscles was very similar to that found by Schwartz et a l .  Similar results were reported by Grupp et a l .  (1981). Timolol, a 3 adrenergic antagonist, had no effect on the response of p a p i l l a r y muscles or a t r i a to ouabain.  Schwartz et a l .  Grupp et a l . (1981) made similar observations. reported that a second dose response  (1981) and  Grupp et a l . (1981)  curve in p a p i l l a r y muscles  displayed only a single component and suggested that this was a result of desensitization. did not f a l l  During the washout period the tension  to predrug levels and i t is possible that the  investigators' f a i l u r e to observe the low-dose effects was due to the fact that the effect was not f u l l y washed out and was s t i l l present when the second dose response curve was performed.  Schwartz  et a l . (1981) proposed that the high a f f i n i t y response was mediated by the action of ouabain on a s i t e unrelated to (Na + K )-ATPase +  +  and that binding to this s i t e resulted in a d i r e c t increase in the i n t r a c e l l u l a r calcium concentration. 79  They suggested that the low  a f f i n i t y s i t e in ventricle  (and the s i t e in atria) was (Na + K ) +  +  ATPase and that ouabain binding inhibited the enzyme and eventually led to an increase  in i n t r a c e l l u l a r calcium.  The f i n a l common step  in both mechanisms of inotropy was the increase  in i n t r a c e l l u l a r  calcium which would then be available to interact with the elements.  contractile  At this time i t appears that there are two ouabain binding  s i t e s in rat ventricular t i s s u e .  It is not clear whether both of  these sites are related to the sodium pump.  Equally unclear is  the  role of this second s i t e in tissues displaying a monophasic response to ouabain.  Schwartz et a l . (1981) suggested that this s i t e may be  present in e l l  tissues with i t s a f f i n i t y being very s i m i l a r to that +  of the s i t e on (Na  +  + K )-ATPase in tissues displaying a monophasic  response to cardiac glycosides.  At this time the identity of this  s i t e as well as its d i s t r i b u t i o n remains unclear. The other variable which we examined was time.  It has been  reported that the response of isolated cardiac tissues to ouabain is dependent on the period of e q u i l i b r a t i o n .  Carrier et a l . (1974)  reported that the response of guinea pig a t r i a to ouabain was enhanced in tissues equilibrated for five hours compared to those equilibrated for only one hour.  The authors actually observed a  decrease in s y s t o l i c tension during the longer equilibration period and although ouabain caused a greater increase  in tension in these  tissues the maximum tension developed did'hotvappear'to  be  different  from that developed by tissues equilibrated for only one hour. did not observe any difference  We  in predrug s y s t o l i c tensions in a t r i a  or p a p i l l a r y muscles equilibrated for one or three hours.  The response  of p a p i l l a r y muscles to cardiac glycosides in the present studies was 80  enhanced by long periods of e q u i l i b r a t i o n .  Care should therefore  be taken to employ the same e q u i l i b r a t i o n time prior to a l l ouabain dose response curves.  The increased e q u i l i b r a t i o n period had a  much less dramatic effect on the response  of a t r i a to ouabain.  It  is possible that the p a p i l l a r y muscles became s l i g h t l y hypoxic over the long e q u i l i b r a t i o n period despite oxygenation of the buffer.  V. 1.  Conclusions S t a t i s t i c a l l y s i g n i f i c a n t differences  in inotropic and chronotro-  pic responses to d,l isoproterenol could not be detected in isolated cardiac tissues from 7 or 70 day diabetic rats as compared to controls. A trend toward a decrease in inotropic and chronotropic response was observed in tissues 70 days after the induction of 2.  diabetes.  Isolated cardiac tissues from chronically diabetic rats had a  decreased capacity to respond to cardiac glycosides.  The effect was  not evident in tissues from acutely diabetic (7 day) r a t s . 3.  Ouabain produced a monophasic response in l e f t a t r i a and a  biphasic response in l e f t papillary muscles. mediated by catecholamine 4.  The response was not  release.  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